Mitochondrial Genome Characteristics Reveal Evolution of Acanthopsetta nadeshnyi (Jordan and Starks, 1904) and Phylogenetic Relationships

In the present study, the mitochondrial genomic characteristics of Acanthopsetta nadeshnyi have been reported and have depicted the phylogenetic relationship among Pleuronectidae. Combined with a comparative analysis of 13 PCGs, the TN93 model was used to review the neutral evolution and habitat evolution catalysis of the mitogenome to verify the distancing and purification selectivity of the mitogenome in Pleuronectidae. At the same time, a species differentiation and classification model based on mitogenome analysis data was established. This study is expected to provide a new perspective on the phylogenetic relationship and taxonomic status of A. nadeshnyi and lay a foundation for further exploration of environmental and biological evolutionary mechanisms.


Introduction
The data analysis of mitogenome is a fundamental indicator for analyzing the relationship between vertebrate evolution and phylogeny [1].In recent years, these studies have provided a clearer positioning of species status on the ecological chain as more and more complete mitochondrial genome information is being reported [2,3].Generally, the mitogenome structure of vertebrates is arranged in a specific order [4].These components include 13 protein-coding genes (ATP6, ATP8, COX1-3, CytB, NAD1-6, and NAD4L), 2 ribosomal ribonucleic acids (rrnS and rrnL), and 22 transfer RNA genes (tRNAs) [5].These characteristic information contain crucial information for molecular evolution, such as base composition bias, codon usage, and substitution rate [6].In addition, the mitogenome with simple structures and low recombination levels exhibits more gene copies with fewer bases, making it widely used in the study of biological evolutionary origins and genetic diversity [7,8].
A. nadeshnyi (Jordan and Starks, 1904) (Acanthopsetta, Pleuronectiformes) is a species of ray-finned fish in the family of right-eye flounders.It is distributed in the northwest Pacific region, including the Korean Peninsula, Japan, and the Bering Sea [9,10].In Pleuronectiformes, there are many species, but the genetic information of the reported species is limited.Several studies on comparative genomics are not sufficient to clarify the complete phylogenetic relationships [11].Therefore, the ecological location of the species is not clear and there is an urgent need to carry out the ecological positioning of A. nadeshnyi.
With the development of molecular sequencing, comparative genomic results can no longer be a single indicator of species classification, and more reports will apply evolutionary data theory to the biological status of species [2,12,13].In previous studies, different gene fragments have created different trees, but the complete evolutionary classification of this species is not clear: Studies have constructed phylogenetic trees for COX1 and 16S rRNA of 10 species of Pleuronectiformes, but these two trees are not completely identical [14]; another study used the control region of the mitogenome as a sample to compare a different tree [15].Ultimately, the incomplete mitogenome cannot exhibit consistent phylogenetic outcomes.A study reported the full length of the mitogenome sequences in A. nadeshnyi, but gene structure and evolutionary intervals have not been analyzed in conjunction with evolutionary trees unfortunately [16].This situation leads to incomplete phylogenetic research and unclear ecological location of A. nadeshnyi.At the same time, the composition characteristics of the genome and the evolutionary trends of related species were analyzed, which will help to locate the ecological position of the species.In order to clarify the evolutionary relationships and states of species, we sequenced and assembled the entire mitochondrial gene of A. nadeshnyi.For the first time, the test of evolutionary divergence time provides a clearer elucidation of A. nadeshnyis' phylogenetic relationship and taxonomic status in this study.

Sample Collection, DNA Extraction, and PCR Amplification and Sequencing
The specimen of A. nadeshnyi was obtained from the east side of the Yellow Sea (33 • 47 ′ 10 ′′ N, 126 • 59 ′ 30 ′′ E) on 15 August 2017.This specimen (carcass length: 32 cm, female) is deposited in the Laboratory of the Museum of Materials and Environmental Engineering (Zhaowen Liu, liuzhaowen92@163.com),under voucher number AN2109073.The total genomic DNA was extracted using a modified phenol-chloroform method from the muscle tissue [3].The sample was subjected to the Sanger sequencing method (Sangon Biotech, Shanghai, China), forming a circular mitochondrial genome.Design primers were based on published mitochondrial genomes and strictly amplified according to the requirements of the reagent kit (Takara, Beijing, China) (PrimeSTAR ® Max DNA Polymerase) (Supplementary Table S1).

Sequence Analysis and Assembly and Mitochondrial Genome Annotation
The sequencing fragment was spliced into a complete circular DNA using the Codon-Code Aligner 5.1.5(CodonCode Corporation, Dedham, MA, USA).We annotated the complete genome using a MITOS online server and manually corrected the tRNA structure (Supplementary Figure S1) [17].

Amino Acid Composition and Nucleotide Substitution Saturation Index of PCGs
The relative synonymous codon usage (RSCU) values and codon numbers were calculated by MEGA 11 [18].The nonsynonymous mutation rates (Ka), synonymous mutation rates (Ks), and Ka/Ks ratio for the PCGs were calculated in DnaSP 5 [19].The nucleotide substitution saturation index of PCGs was calculated using DAMBE, and the substitution fitting model for the three codons was TN93 [20].

Relative Evolutionary Rate Analysis
Mitochondrial genomes from 25 species were aligned using MUSCLE v3.8.31 (https://drive5.com/muscle/,accessed on 23 March 2024) and concatenated into a supergene for each species.Evolutionary rates were determined from these super-gene sets using the tpcv module in the LINTRE program [21].

Divergence Time Estimation
The divergence times were estimated based on Beast 2 [22] via the approximate likelihood calculation method of the MCMCtree program, with the following parameters: --Substitution Model GTR; --Site Heterogeneity Model γ; --Tree Prior Yule Process; --Length of chain 10,000,000; and --Echo state to screen every 1000.Fossil records acquired from the TimeTree website (http://www.timetree.org)were used to calibrate the divergence times.We used TVBOT as a graphic beautification tool (https://www.chiplot.online/tvbot.html,accessed on 25 March 2024) [23].

Protein-Coding Genes and Codon Usage
The total length of all PCGs was 10596 bp in the mitogenome of A. nadeshnyi, which accounted for 61.57% of the whole genome (Table 2).The comparison of the initiation of all PCGs showed that all CDs start with ATG as the starting codon, except for COX1 which was GTG.In terms of terminating codons, each CD was different: the COX2, ND3, ND4, In the mitogenomes, most of the coding fragments were on the heavy strand.Among them, eight tRNAs (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro) were on the light strand, and the other fourteen tRNAs were all on the heavy strand.The length of each tRNA ranged from 65 to 74 bp (Table 2), and they were able to form a stable clover structure (Supplementary Figure S1).The small coding subunit (12S rRNA) and large coding subunit (16S rRNA) appeared on both sides with tRNA-Phe and tRNA-Leu, which were located on the H-chain and separated by the tRNA-Val.They were 950 bp and 1714 bp lengths, respectively (Table 2).Except for ND6, all remaining CD areas were located on the heavy strand (Figure 1).

Protein-Coding Genes and Codon Usage
The total length of all PCGs was 10596 bp in the mitogenome of A. nadeshnyi, which accounted for 61.57% of the whole genome (Table 2).The comparison of the initiation of all PCGs showed that all CDs start with ATG as the starting codon, except for COX1 which was GTG.In terms of terminating codons, each CD was different: the COX2, ND3, ND4, and Cytb genes used an incomplete T stop codon; the COX3 gene used TA; the ND2 gene used TAG; and the ND1, COX1, ATP8, ATP6, ND4L, ND5, and ND6 genes used TAA.
In an RSCU analysis, different codon usage frequencies indicated the selection evolutionary pressure of different amino acids (Figure 2).Generally speaking, the codon composition of longer amino acids is more abundant [30].In this study, Leu1, Ser 2, Pro, and Thr also showed greater abundance compared to other amino acids.Interestingly, Val, Arg, and Gly exhibited a higher abundance with fewer quantities.Therefore, among multiple amino acid frequencies, Val, Arg, and Gly might also have a more stable genetic efficiency [31].On the other hand, other amino acids may undergo relatively unstable genetic evolution due to genetic mutations or random genetic drift.These unstable sites may choose better amino acid codons due to different environmental selection pressures [30].
In an RSCU analysis, different codon usage frequencies indicated the selection evolutionary pressure of different amino acids (Figure 2).Generally speaking, the codon composition of longer amino acids is more abundant [30].In this study, Leu1, Ser 2, Pro, and Thr also showed greater abundance compared to other amino acids.Interestingly, Val, Arg, and Gly exhibited a higher abundance with fewer quantities.Therefore, among multiple amino acid frequencies, Val, Arg, and Gly might also have a more stable genetic efficiency [31].On the other hand, other amino acids may undergo relatively unstable genetic evolution due to genetic mutations or random genetic drift.These unstable sites may choose better amino acid codons due to different environmental selection pressures [30].

Mitogenome Mutations and Evolutionary Relationships in Pleuronectidae
In the publicly available mitogenomes, we selected twenty-three typical genomes of Pleuronectidae for calculating the evolutionary selection pressure of A. nadeshnyi.Meanwhile, we set up species A. dabryanus with the same close relationship and species

Mitogenome Mutations and Evolutionary Relationships in Pleuronectidae
In the publicly available mitogenomes, we selected twenty-three typical genomes of Pleuronectidae for calculating the evolutionary selection pressure of A. nadeshnyi.Meanwhile, we set up species A. dabryanus with the same close relationship and species E. pelecanoides and S. lavenbergi with two distant relationships to verify the accuracy of the results mutually.Based on the gene sequences of 13 PCGs of the mitogenomes, the relative evolutionary pressure of each species could be characterized.As shown in the results (Figure 3A), the evolutionary pressure on E. jordani and E. pelecanoides was relatively low.In other species, most stress indices were around −0.15, and this might be related to the genetic stability of mitochondria [32].The relatively similar mutation pressure index not only demonstrates the maternal heritability of the mitogenomes but may also indicate the similarity of environmental selection pressure [33].In the analysis of synonymous mutations in amino acids, different Ka and Ks values exhibit relatively similar Ka/Ks values (Figure 3B).The difference in Ka values among different Pleuronectidae species was relatively small, which may indicate that the frequency of neutral evolution was relatively similar among different species, and this accumulation of neutral evolution may also lead to non-environmental selectivity of the mitogenomes [34].In the evolutionary process of the Pleuronectidae species, constrained evolution and divergent evolution seemed to be closely related to gene mutations (Ka/Ks).In most evolution, evolutionary selection of the mitogenomes eliminates harmful gene mutations and maintains the stability of the original amino acids [35].
Genes 2024, 15, x FOR PEER REVIEW 7 of 12 E. pelecanoides and S. lavenbergi with two distant relationships to verify the accuracy of the results mutually.Based on the gene sequences of 13 PCGs of the mitogenomes, the relative evolutionary pressure of each species could be characterized.As shown in the results (Figure 3A), the evolutionary pressure on E. jordani and E. pelecanoides was relatively low.
In other species, most stress indices were around −0.15, and this might be related to the genetic stability of mitochondria [32].The relatively similar mutation pressure index not only demonstrates the maternal heritability of the mitogenomes but may also indicate the similarity of environmental selection pressure [33].In the analysis of synonymous mutations in amino acids, different Ka and Ks values exhibit relatively similar Ka/Ks values (Figure 3B).The difference in Ka values among different Pleuronectidae species was relatively small, which may indicate that the frequency of neutral evolution was relatively similar among different species, and this accumulation of neutral evolution may also lead to non-environmental selectivity of the mitogenomes [34].In the evolutionary process of the Pleuronectidae species, constrained evolution and divergent evolution seemed to be closely related to gene mutations (Ka/Ks).In most evolution, evolutionary selection of the mitogenomes eliminates harmful gene mutations and maintains the stability of the original amino acids [35].At the same time, we used TN93 as a model to verify the base substitution ratio and nucleotide frequency of the three codons (Figure 4) [36].Generally speaking, the nonsynonymy of the second codon (Figure 4B) and the synonymy of the third codon (Figure 4C) jointly affect changes in amino acid evolution [37].In the results of this study, Figure 4B,C exhibited different enrichment patterns; the common frequency of the first and second codons (Figure 4D) was similar to the frequency characteristics of the first codon (Figure 5), and nonsynonymous mutations may approach neutral mutations.This result At the same time, we used TN93 as a model to verify the base substitution ratio and nucleotide frequency of the three codons (Figure 4) [36].Generally speaking, the nonsynonymy of the second codon (Figure 4B) and the synonymy of the third codon (Figure 4C) jointly affect changes in amino acid evolution [37].In the results of this study, Figure 4B,C exhibited different enrichment patterns; the common frequency of the first and second codons (Figure 4D) was similar to the frequency characteristics of the first codon (Figure 5), and nonsynonymous mutations may approach neutral mutations.This result was mutually consistent with the above inference (Ka/Ks) and was also supported by the purify selection theory [38].
Genes 2024, 15, x FOR PEER REVIEW 8 of 12 was mutually consistent with the above inference (Ka/Ks) and was also supported by the purify selection theory [38].was mutually consistent with the above inference (Ka/Ks) and was also supported by the purify selection theory [38].The life evolution scale of this chart is based on the relative divergence time of two outer groups E. pelecanoides and S. lavenbergi as a unit.Among them, the divergence time between E. pelecanoides and S. lavenbergi is 25.9-118.3Mya, and 39 Mya is selected as the unit scale based on species affinity (https://timetree.org).

Divergence Time and Phylogenetic Analysis
In recent years, divergence time estimation has become increasingly prominent in evolutionary biology [39].Methodological and empirical advances now allow time trees to be estimated more accurately than ever before [26].It is assumed that the molecular evolution rate of a species is approximately constant, that is, the evolution rate of genetic differences should be proportional to the time of differentiation (molecular clock) [40].For any large molecule (DNA sequence or protein sequence), there is an approximately constant evolutionary rate across all evolutionary lineages [41].If the number of mutations aggregated on an evolutionary branch is proportional to the length of independent evolution time of that branch, then its replacement rate may approximately maintain a constant value during the evolution process [42].Generally speaking, an accurate estimation method is to use the fossil time of a specific group as a correction, and then estimate the divergence time between species based on the degree of divergence between gene sequences and molecular clocks [43].We can simultaneously estimate the occurrence time of other nodes on the phylogenetic tree in order to infer the origin of related groups and the divergence time of different groups [41,43].
In this study, all PCGs of the mitochondrial basic groups of twenty-seven species were used as units for calculating the evolutionary time tree (Figure 5).The life evolution scale of Figure 5 is based on the relative divergence time of two outer groups E. pelecanoides and S. lavenbergi as a unit.Among them, the divergence time between E. pelecanoides and S. lavenbergi is 25.9-118.3Mya, and 39 Mya is selected as the optimal unit scale based on species affinity (https//timetree.org).It was interesting that species under the same subject cluster to other subjects.The clustering of most species of Pleuronectidae was natural, except for A. stomias and E. jordani.In the topology of divergence time (Figure 5), the separation time of A. dabryanus (Acipenseridae) was approximately 297.78-340.32Mya, but it clustered together with species from other families.This may be convergent evolution of A. dabryanus and P. cornutus or other species of Pleuronectidae in similar habitats [44].Perhaps due to the environment at different latitudes, light, and temperature, A. stomias and E. jordani underwent a divergent evolutionary process.Unfortunately, the comparison of mitochondrial genomes alone cannot be the sole explanation for this phenomenon, and it was necessary to correctly verify the survival range of all species worldwide.
Except for the divergence time of the cross-clustering, all species exhibited consistency in divergence time and sequence tree construction (Figure 5) (Supplementary Figure S2).The tree construction of the two results showed almost similar clustering topology.A. nadeshnyi and D. rikuzenius differentiated over a period of 42.54 Mya during the Cenozoic (Figure 5), and similar conclusions were also reflected in the results of systematic development (Supplementary Figure S2).The differentiation time of Pleuronectidae was 42.54-340.32Mya, and this may be related to the changes in Quaternary glacier movement [45][46][47].Unfortunately, due to the ancient nature of the mitogenome, the results of gene tree construction may be different from those of the species tree.

Conclusions
In this study, we reported the complete mitochondrial genome of A. nadeshnyi, analyzed the corresponding genomic information, compared with the reported mitogenomes of A. nadeshnyi and congeneric species, and depicted the phylogenetic relationship among Pleuronectidae.Meanwhile, combined with a comparative analysis of 13 PCGs, the TN93 model was used to review the neutral evolution and environmental evolution catalysis of the mitochondrial genome to verify the distancing and purification selectivity of the mitochondrial genome in Pleuronectidae.A cross-analysis model for species differentiation and tracing was established using mitochondrial genome data using divergence time and phylogenetic analysis.In future research, more mitochondrial genome data will be made public, and this data model will be more accurate when combined with the historical changes in the coastline.This study provides basic data support for analyzing the ge-

Figure 2 .
Figure 2. The relative synonymous codon usage (RSCU) in the mitogenome of A. nadeshnyi (A).(The y-axis represents the usage frequency of the corresponding amino acid codons in 13 PCGs.Different colors represent the different codons in the amino acids.)The amino acid composition in the mitogenome of A. nadeshnyi (B).(The x-and y-axis refer to the amino acid composition and the number of each amino acid in 13 PCGs, respectively.)

Figure 2 .
Figure 2. The relative synonymous codon usage (RSCU) in the mitogenome of A. nadeshnyi (A).(The y-axis represents the usage frequency of the corresponding amino acid codons in 13 PCGs.Different colors represent the different codons in the amino acids.)The amino acid composition in the mitogenome of A. nadeshnyi (B).(The x-and y-axis refer to the amino acid composition and the number of each amino acid in 13 PCGs, respectively.)

Figure 3 .
Figure 3.The relative evolutionary pressure index of species based on the mitochondrial genome model of Pleuronectidae (A).The ratio (Ks/Ks) of synonymous substitution (Ka) and synonymous substitution (Ks) calculated using amino acids as data points indicates the mutation pressure index of the mitochondrial genome (B).

Figure 3 .
Figure 3.The relative evolutionary pressure index of species based on the mitochondrial genome model of Pleuronectidae (A).The ratio (Ks/Ks) of synonymous substitution (Ka) and synonymous substitution (Ks) calculated using amino acids as data points indicates the mutation pressure index of the mitochondrial genome (B).

Figure 4 .
Figure 4.Nucleotide substitution saturation plots for all 13 PCGs of Pleuronectidae.First codon positions (A); second codon positions (B); third codon positions (C); and first codon and second codon positions (D).Plots in blue and green indicate transition and transversion, respectively.

Figure 5 .
Figure 5.The divergence time and geological scale of species mitochondrial genomes of Pleuronectidae.The life evolution scale of this chart is based on the relative divergence time of two outer groups E. pelecanoides and S. lavenbergi as a unit.Among them, the divergence time between E. pelecanoides and S. lavenbergi is 25.9-118.3Mya, and 39 Mya is selected as the unit scale based on species affinity (https://timetree.org).

Figure 4 .
Figure 4.Nucleotide substitution saturation plots for all 13 PCGs of Pleuronectidae.First codon positions (A); second codon positions (B); third codon positions (C); and first codon and second codon positions (D).Plots in blue and green indicate transition and transversion, respectively.

Figure 4 .
Figure 4.Nucleotide substitution saturation plots for all 13 PCGs of Pleuronectidae.First codon positions (A); second codon positions (B); third codon positions (C); and first codon and second codon positions (D).Plots in blue and green indicate transition and transversion, respectively.

Figure 5 .
Figure 5.The divergence time and geological scale of species mitochondrial genomes of Pleuronectidae.The life evolution scale of this chart is based on the relative divergence time of two outer groups E. pelecanoides and S. lavenbergi as a unit.Among them, the divergence time between E. pelecanoides and S. lavenbergi is 25.9-118.3Mya, and 39 Mya is selected as the unit scale based on species affinity (https://timetree.org).

Figure 5 .
Figure 5.The divergence time and geological scale of species mitochondrial genomes of Pleuronectidae.The life evolution scale of this chart is based on the relative divergence time of two outer groups E. pelecanoides and S. lavenbergi as a unit.Among them, the divergence time between E. pelecanoides and S. lavenbergi is 25.9-118.3Mya, and 39 Mya is selected as the unit scale based on species affinity (https://timetree.org).

Table 1 .
Species attribution and accession number.

Table 2 .
Features of mitochondrial genomes of A. nadeshnyi.
* Intergenic region: non-coding bases between the feature on the same line and the line below, with a negative number indicating an overlap.# H: heavy strand; L: light strand.